STRUCTURE/FUNCTION STUDIES ON METALLO-?-LACTAMASE ImiS FROM
Aeromonas bv. sobria
by Narayan Prasad Sharma
Zinc-containing metallo-?-lactamases(M?Ls) are an emerging class of enzymes
that render bacteria resistant to ?-lactam-containing antibiotics, and these enzymes have
been grouped into 3 distinct classes, B1, B2 and B3. While extensive
structure/mechanistic information is known about the B1 and B3 M?Ls, relatively little is
known about the B2 enzymes. In an effort to characterize a B2 M?L, spectroscopic and
mechanistic studies on metallo-?-lactamase ImiS from Aeromonas bv. sobria, were
performed. 1H NMR, UV-Vis, EPR, and EXAFS spectroscopic studies on Co(II)substituted
ImiS revealed that the active site metal ion is tetrahedrally-coordinated by 1
cysteine, 1 histidine, and 1 aspartic acid and presumably 1 water ligands. Steady-state,
presteady-state kinetic studies were conducted on ImiS and its reaction with imipenem
and meropenem. pH Dependence studies revealed no inflection points in the pH range
5.0-8.5, while proton inventories demonstrated at least 1 rate-limiting proton transfer.
Stopped-flow fluorescence, stopped-flow UV-Vis, and rapid freeze quench EPR studies
on Co(II)-substituted ImiS revealed a kinetic mechanism in which the rate-limiting step
is C-N bond cleavage. Site-directed spin labeling and EPR spectroscopy have revealed
the movement of ?-helix in kinetically competent time scale. Taken together, this
dissertation offers, the only structural and mechanistic information on a B2 M?L and a
common rate-limiting step for all M?L’s that can be used to guide future inhibitor design
efforts.